Many enveloped viruses contain an internal matrix (M) protein as part of the viral envelope. The M protein plays a central role in virus assembly by binding the nucleoprotein core (nucleocapsid to the cytoplasmic surface of the plasma membrane during the budding process. The objective of the proposed experiments is to determine the mechanisms by which the M protein of vesicular stomatitis virus (VSV) binds the viral nucleocapsid and envelope glycoprotein (G protein) in virus assembly. The approach is to use novel biochemical and biophysical assays for viral protein interactions in vitro correlated with results of genetic manipulation of viral protein function in vivo. The first Specific Aim is to determine the factors that induce M protein binding to nucleocapsids. In vitro assays for M protein binding to nucleocapsids will be used to characterize the effects of mutations in the M protein on virus assembly in vivo. The questions to be addressed include the nature of the steps involved in initiation of assembly and the role of different M protein subpopulations in inducing condensation of the nucleocapsid into the tightly coiled, bullet-shaped structure found in virions. The second Specific Aim is to determine the role of M protein phosphorylation in virus assembly. This will be accomplished by characterizing the function of M proteins containing mutations that abolish the ability of M protein to be phosphorylated. The third Specific Aim is to determine the mechanism of binding of the M protein to the G protein. A recently developed binding assay for the interaction of M protein with G .protein in vitro will be used to determine the structural features of these proteins. involved in binding. The binding of M protein to other viral envelope glycoproteins will be studied to determine the mechanisms involved in phenotypic mixing of viral envelope proteins. The experiments are designed to address questions about virus assembly that have existed for many years. These include the nature of the signals that initiate virus assembly at the plasma membrane, the mechanism by which the M protein provides the virions with its distinctive morphology, the basis of phenotypic mixing of viral envelope glycoproteins, and the mechanism of the often hypothesized but elusive interaction between viral envelope and matrix proteins.